Enterobacteriaceae are a common cause of healthcare-associated bacterial infections, including pneumonia, meningitis, sepsis, and other life threatening illness, especially among patients with underlying medical conditions. The recent rise of carbapenem-resistant Enterobacteriaceae (CRE) has left clinicians with limited antimicrobial treatment options for these infections, and has been declared an immediate public health threat that requires urgent and aggressive action by the Centers for Disease Control and Prevention [1]. Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae are now one of the most widely disseminated CRE pathogens, and are associated with high morbidity and mortality rates [2, 3]. Since their initial identification in 2001 [1], KPC-producing K. pneumoniae have emerged throughout the United States (currently identified in 47 states; CDC unpublished data) and the world, spanning five continents that also include South America, Eurasia, Africa and Australia [5-7].
The rapid, widespread dissemination of KPC-producing K. pneumoniae is largely attributed to the clonal expansion of a single dominant strain, sequence type (ST) 258 as defined by multilocus sequence typing or MLST, currently circulating in over 20 countries [6]. ST258 is a member of the recently designated clonal group (CG) 258 [8], which comprises several other sequence types linked to outbreaks, suggesting that these strains may share genetic features that predispose them to pathogenicity or increased transmissibility. Unlike ST258, other CG258 strains are associated with a variety of carbapenemases including KPC, NDM, VIM, and OXA-48 [9-11]. The transmission of KPC-producing ST258 and other CG258 strains is frequently linked to patient travel or healthcare exposure in known endemic areas, such as the United States, Israel, and Greece [6, 12, 13]. Despite previous genomic analyses of ST258 [14-18], an explanation for its pathogenic success in the healthcare system remains unclear.
Large homologous recombination events frequently shape genomes to result in new emerging pathogens [19]. A sequence of these events has now been documented for CG258 and ST258. Gaiarsa and colleagues, using sequence from Italian isolates and the public database, discovered a putative recombination event that gave rise to CG258. Their evidence shows a donor related to K. pneumoniae ST1628 contributed ˜1.3 Mbp to an ancestor of ST11 (CG258) sometime before 1985 [17]. Chen and colleagues used public genomic data to show the ST258 lineage resulted from a ˜1.1 Mbp recombination event between ST11 and a strain related to a Brazilian ST442 isolate [20]. DeLeo and colleagues published a whole genome SNP-based phylogeny of ST258 from mostly the northeastern U.S., and concluded that ST258 comprises two distinct lineages which diverged after a homologous recombination event of ˜215 kb that included the capsule polysaccharide synthesis (cps) locus [15]. Additionally, Wyres and colleagues documented several recombination events involving cps loci in CG258 [18].
Given these concerns and motivations, there is a demonstrated need in the art for a panel of real-time PCR assays, based on competitive probe methodology, for rapid and sensitive molecular detection of clinically relevant Klebsiella pneumoniae strains. Competitive probe assays, a form of allele-specific PCR (ASPCR), employs primers that are designed for SNP analysis.